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Plus-end and minus-end directed motors are associated with specialized Sertoli cell junction plaques (ectoplasmic specializations) : evidence from an in vitro polarity marked microtubule motility assay

University of British Columbia

Developing sperm cells (spermatids) translocate within the seminiferous epithelium during spermatogenesis. It has been proposed that this translocation results from the microtubule-based transport of specialized junction plaques that occur in Sertoli cells in regions where these cells are attached to spermatids. These junction plaques each consist of a cistern of endoplasmic reticulum, a layer of actin filaments, and a region of the plasma membrane involved with intercellular adhesion. Molecular motors anchored to the cytoplasmic face of the endoplasmic reticulum are thought to move the junction plaques, and hence the attached spermatids, towards the base and back to the apex of the epithelium, along microtubules. In Sertoli cells, microtubules are arranged parallel to the long axis of the cell and have their minus ends located at the apex of the epithelium. If the spermatid translocation hypothesis is true, then isolated junction plaques should support microtubule movement both in the plus and minus direction when tested in vitro. To verify this prediction, I have developed and have used an in vitro polarity-marked microtubule motility assay. Polarity labeled microtubules were bound to isolated spermatid/junction complexes and then the complexes were added to motility chambers. The complexes then were assessed for their ability to transport microtubules in the presence of a motility buffer containing ATP and testicular cytosol (2 mg/ml). The presence of junction plaques attached to spermatids was established with fluorescently labeled phallotoxin for filamentous actin, and with DIOC6 for endoplasmic reticulum. Of 25 recorded motility events, 17 were in the plus-end direction and 8 were in the minusend direction (on a bed of purified kinesin 35/37 polar microtubules moved in the plusend direction and on a bed of testicular cytosol all 158 polar microtubules moved in the plus-end direction). The results are consistent with the conclusion that the junction plaques have the potential for moving along microtubules in both the plus and minus directions. Also, together with some of my data from immunofluoresence studies, they constitute the first evidence that a kinesin-type motor is associated with the junction plaques at stages when translocation occurs in vivo.

Thesis/Dissertation

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2009-06-26

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http://hdl.handle.net/2429/9680

Anatomy, Cell Biology and Physiology

University of British Columbia

UBCV

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